The present invention relates to compositions for eliciting an immunological response in a host, animal or human, and methods for making and using the same. The invention further relates to such compositions and methods wherein the composition comprises an antigen and a lipoprotein adsorbed to an adjuvant. More preferably, the lipoprotein is also antigenic or immunogenic, and thus the composition can be a combination, multivalent or xe2x80x9ccocktailxe2x80x9d composition. Accordingly, the invention also relates to co-administration of at least one antigen and at least one lipoprotein in a composition which can include additional ingredients, such as an adjuvant.
The lipoprotein can be a naturally occurring lipoprotein or a recombinant lipoprotein. The recombinant lipoprotein can be from expression by a vector of homologous sequences for the lipidated and protein portions of the lipoprotein, i.e., the sequences for the lipidation and protein can naturally occur together. In such a recombinant lipoprotein, the lipidation thereof can be from expression of a first nucleic acid sequence and the protein thereof can be from expression of a second nucleic acid sequence, wherein the first and second nucleic acid sequences, which do not naturally occur together, and such sequences can be expressed as a contiguous lipoprotein. Thus, the invention relates to compositions and methods involving administration of lipoproteins, including recombinant lipoproteins; and the recombinant lipoproteins can be similar to native proteins, or novel hybrid proteins.
The invention further relates to the aforementioned compositions for eliciting an immunological response and methods for making and using the same wherein the lipoprotein is recombinantly expressed lipoprotein from expression of such aforementioned first and second nucleic acid sequences wherein the first nucleic acid sequence encodes a Borrelia lipoprotein leader sequence; preferably such a recombinant lipidated protein expressed using the nucleic acid sequence encoding the OspA leader sequence. In a preferred embodiment the lipoprotein can be OspA; and thus, the invention also relates to recombinant OspA and uses thereof the compositions and methods.
Several publications are referenced in this application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned; and each of these publications is hereby incorporated herein by reference.
Immunogenicity can be significantly improved if an antigen is co-administered with an adjuvant, commonly used as 0.001% to 50% solution in phosphate buffered saline (PBS). Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune response to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccarides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune response. Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.
A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund""s complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. To efficiently induce humoral immune response (HIR) and cell-mediated immunity (CMI), immunogens are preferably emulsified in adjuvants.
Desirable characteristics of ideal adjuvants include any or all of:
(1) lack of toxicity;
(2) ability to stimulate a long-lasting immune response;
(3) simplicity of manufacture and stability in long-term storage;
(4) ability to elicit both CMI and HIR to antigens administered by various routes;
(5) synergy with other adjuvants;
(6) capability of selectively interacting with populations of antigen presenting cells (APC);
(7) ability to specifically elicit appropriate TH1 or TH2 cell-specific immune responses; and
(8) ability to selectively increase appropriate antibody isotype levels (for example IgA) against antigens.
U.S. Pat. No. 4,855,283 granted to Lockhoff et al. on Aug. 8, 1989 which is incorporated herein by reference thereto teaches glycolipid analogs including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immune-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No. 4,855,283) reported that N-glycolipids analogs displaying structural similarities to the naturally occurring glycolipids, such as glycosphingolipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain alkylamines and fatty acids that are linked directly with the sugar through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.
U.S. Pat. No. 4,258,029 granted to Moloney, assigned to Connaught Laboratories Limited and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Octodecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen, enhanced the host immune responses against hepatitis B virus.
Bessler et al., xe2x80x9cSynthetic lipopeptides as novel adjuvants,xe2x80x9d in the 44th Forum In Immunology (1992) at page 548 et seq., especially at 548-550, incorporated herein by reference, is directed to employing lipopeptides as adjuvants when given in combination with an antigen. The lipopeptides typically had P3C as the lipidated moiety and up to only 5 amino acids, e.g., P3C-SG, P3C-SK4, P3C-SS, P3C-SSNA, P3C-SSNA. The lipopeptide was coupled with or added to only certain antigens or to non-immunogenic proteins, such as P3C-SSNA supplementing S. typhimurium vaccine, PC3-SS coupled to VP1(135-154) of foot-and-mouth disease, PC3-SG-OSu coupled to non-immunogenic protein hirudin, P3C-SK coupled to FITC or DNP or P3C-SG coupled to a metabolite from Streptomyces venezuelae. While adjuvant mixing and conjugating procedures of Bessler can be employed in the practice of the present invention, Bessler fails to teach or suggest employing a lipoprotein with at least one antigen in a composition, especially such compositions wherein the lipoprotein is also antigenic, or the immunological combination compositions and methods of this invention.
In this regard, a distinction between a peptide, especially a peptide having up to only about 5 amino acids, and a protein is being made, as is a distinction between an antigenic lipoprotein and a non-antigenic lipopeptide, inter alia. Peptides differ immunologically from proteins in that short peptides have the potential for direct presentation by the major histocompatibility complex (MHC), while proteins require processing prior to presentation to T-cells. A peptide further differs from a protein in that a protein is large enough that it is capable of forming functional domains (i.e., having tertiary structure), whereas a peptide cannot.
Nardelli et al. [Vaccine (1994), 12(14):1335-1339] covalently linked a tetravalent multiple antigen peptide containing a gp120 sequence to a lipid moiety and orally administered the resulting synthetic lipopeptide to mice. It was found that both mucosal IgA response and systemic plasma IgG were stimulated, and cell-mediated immunity, as shown by lymphokine production and generation of a specific cytotoxic response, was induced. Only a short peptide was used, rather than a whole lipoprotein, and there is no teaching or suggestion that the synthetic lipopeptide could be used as an adjuvant for other proteins. In fact, this reference actually teaches away from the use of lipoproteins, which are more soluble than lipopeptides, as immunogens; see, e.g. p. 1338, last line (xe2x80x9csoluble proteins are not immunogens by oral routesxe2x80x9d).
Croft et al. [J. Immunol. (1991), 146(5): 793-796] have covalently coupled integral membrane proteins (Imps) isolated from E. coli to various antigens and obtained enhanced immune responses by intramuscular injection into mice and rabbits. However, there are disadvantages to coupling the lipoprotein and the antigen covalently. Important epitopes may be damaged, and the coupling procedure is difficult to control and often requires the use of toxic cross-linkers. Thus, it would be advantageous to provide a method for inducing an enhanced immunological response which does not require that the antigen be cross-linked to a protein. Moreover, when the antigen CSP-OVA was merely mixed, rather than covalently linked, with the lipoprotein TraT, only a small increase in antibody response was obtained. Croft et al. therefore concluded that the lipid is not necessary for the adjuvant effect, contrary to the surprising findings of the present inventors.
U.S. Pat. No. 4,439,425 relates to lipopeptides having 2 to 10 amino acids and their prophylactic administration by oral or rectal routes.
Bessler et al. [xe2x80x9cSynthetic Lipopeptide Conjugates Constitute Efficient Novel Immunogens and Adjuvants in Parenteral and Oral Immunizationxe2x80x9d (Abstract), Meeting on Molecular Approaches to the Control of Infectious Diseases, (Sep. 13-17, 1995), Cold Spring, Harbor Laboratory (not prior art in view of Jun. 7, 1995 filing date of U.S. Ser. No. 08/476,656)] relates to the oral administration of lipopeptides having six amino acids which were covalently coupled to antigens. The lipopeptide-antigen conjugates were found to induce a hapten-specific immune response.
Schlecht et al. [Zbl. Bakt. (1989) 271:493-500] relates to Salmonella typhimurium vaccines supplemented with synthetically prepared derivatives of a bacterial lipoprotein having five amino acids. The vaccines were administered by two intraperitoneal injections and challenged intraperitoneally with graded doses of S. typhimurium. When the protective capacity of the supplemented vaccines was compared with that of the unsupplemented vaccine, it was found that 90% of the S. typhimurium vaccine could be replaced by the lipopeptide without a recognizable decrease in protective capacity.
Substantial effort has been directed toward the development of a vaccine for Lyme disease. Two distinct approaches have been used for vaccine development. One approach is to use a vaccine composed of whole inactivated spirochetes, as described by Johnson in U.S. Pat. No. 4,721,617. A whole inactivated vaccine has been shown to protect hamsters from challenge and has been licensed for use in dogs.
Due to the concerns about cross-reactive antigens within a whole cell preparation, human vaccine research has focused on the identification and development of non-cross-reactive protective antigens expressed by B. burgdorferi. Several candidate antigens have been identified to date. Much of this effort has focused on the most abundant outer surface protein of B. burgdorferi, namely outer surface protein A (OspA), as described in published PCT patent application WO 92/14488, assigned to the assignee hereof. Several versions of this protein have been shown to induce protective immunity in mouse, hamster and dog challenge studies. Clinical trials in humans have shown the formulations of OspA to be safe and immunogenic in humans [Keller et al., JAMA (1994) 271:1764-1768]. Indeed, one formulation containing recombinant lipidated OspA as described in the aforementioned WO 92/14488, is now undergoing Phase III safety/efficacy trials in humans.
While OspA is expressed in the vast majority of clinical isolates of B. burgdorferi from North America, a different picture has emerged from examination of the clinical Borrelia isolates in Europe. In Europe, Lyme disease is caused by three genospecies of Borrelia, namely B. burgdorferi , B. garinii and B. afzelli. In approximately half of the European isolates, OspA is not the most abundant outer surface protein. A second outer surface protein C (OspC) is the major surface antigen found on these spirochetes. In fact, a number of European clinical isolates that do not express OspA have been identified. Immunization of gerbils and mice with purified recombinant OspC produces protective immunity to B. burgdorferi strains expressing the homologous ospc protein [V. Preac-Mursic et al., INFECTION (1992) 20:342-349; W. S. Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926]. The OspC protein is currently being considered as a possible component of a second generation Lyme vaccine formulation.
Recombinant proteins are promising vaccine or immunogenic composition candidates, because they can be produced at high yield and purity and manipulated to maximize desirable activities and minimize undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or immunogenic compositions. Moreover, it would be greatly desired to be able to administer such proteins in combination with other antigens.
A very promising immune stimulator is the lipid moiety N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine, abbreviated Pam3Cys. This moiety is found at the amino terminus of the bacterial lipoproteins which are synthesized with a signal sequence that specifies lipid attachment and cleavage by signal peptidase II. Synthetic peptides that by themselves are not immunogenic induce a strong antibody response when covalently coupled to Pam3Cys [Bessler et al. (1992)].
In addition to an antibody response, one often needs to induce a cellular immune response, particularly cytoxic T lymphocytes (CTLs). Pam3Cys-coupled synthetic peptides are extremely potent inducers of CTLs, but no one has yet reported CTL induction by large recombinant lipoproteins.
The nucleic acid sequence and encoded amino acid sequence for OspA are known for several B. burgdorferi clinical isolates and is described, for example, in published PCT application WO 90/04411 (Symbicom AB) for B31 strain of B. burgdorferi and in Johnson et al., Infect. Immun. 60:1845-1853 for a comparison of the ospA operons of three B. burgdorferi isolates of different geographic origins, namely B31, ACA1 and Ip90.
As described in WO 90/04411, an analysis of the DNA sequence for the B31 strain shows that the OspA is encoded by an open reading frame of 819 nucleotides starting at position 151 of the DNA sequence and terminating at position 970 of the DNA sequence (see FIG. 1 therein). The first sixteen amino acid residues of OspA constitute a hydrophobic signal sequence of OspA. The primary translation product of the full length B. burgdorferi gene contains a hydrophobic N-terminal signal sequence which is a substrate for the attachment of a diacyl glycerol to the sulfhydryl side chain of the adjacent cysteine residue. Following this attachment, cleavage by signal peptidase II and the attachment of a third fatty acid to the N-terminus occurs. The complete lipid moiety is termed Pam3Cys. It has been shown that lipidation of OspA is necessary for immunogenicity, since OspA lipoprotein with an N-terminal Pam3Cys moiety stimulated a strong antibody response, while OspA lacking the attached lipid did not induce any detectable antibodies [Erdile et al., Infect. Immun., (1993), 61:81-90].
Published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence of the ospc gene of B. burgdorferi strain Pko and the OspC (termed pC in this reference) protein encoded thereby of 22 kDa molecular weight. This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used for OspA. Based on the findings regarding OspA, one might expect that lipidation of recombinant OspC would be useful to enhance its immunogenicity; but, as discussed in above-referenced U.S. Ser. No. 08/475,781, the therein applicants experienced difficulties in obtaining detectable expression of recombinant OspC. It would be useful to enhance the immunogenicity of recombinant OspC. Moreover, it would be useful to have a multivalent Lyme Disease immunological composition which contains antigens against both North American and European Borrelia isolates.
Streptoccus pneumoniae causes more fatal infections world-wide than almost any other pathogen. In the U.S.A., deaths caused by S. pneumoniae rival in numbers those caused by AIDS. Most fatal pneumoccal infections in the U.S.A. occur in individuals over 65 years of age, in whom S. pneumoniae is the most common cause of community-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunodeficient patents including those with sickle cell disease. In the less-developed areas of the world, pneumococcal infection is one of the largest causes of death among children less than 5 years of age. The increase in the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospect for control of pneumococcal disease problematical.
The reservoir of pneumococci that infect man is maintained primarily via nasopharyngeal human carriage. Humans acquire pneumococci first through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain in nasal mucosa for weeks or months. As many as 50% or more of young children and the elderly are colonized. In most cases, this colonization results in no apparent infection. In some individuals, however, the organism carried in the nasopharynx can give rise to symptomatic sinusitis of middle ear infection. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia. Infections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially if they continue to be shed in large numbers from the original focus of infection. Pneumococci in the blood can reach the brain where they can cause meningitis. Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is particularly devastating; some 10% of patients die and greater than 50% of the remainder have life-long neurological sequelae.
In elderly adults, the present 23-valent capsular polysaccharide vaccine is about 60% effective against invasive pneumococcal disease with strains of the capsular types included in the vaccine. The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to make adequate responses to most polysaccharides. Improved vaccines that can protect children and adults against invasive infections with pneumococci would help reduce some of the most deleterious aspects of this disease.
The S. pneumoniae cell surface protein PspA has been demonstrated to be a virulence factor and a protective antigen. In published international patent application WO 92/14488, there are described the DNA sequences for the pspA gene from S. pneumoniae Rx1, the production of a truncated form of PspA by genetic engineering, and the demonstration that such truncated form of PspA confers protection in mice to challenge with live pneumococci.
In an effort to develop a vaccine or immunogenic composition based on PspA, PspA has been recombinantly expressed in E. coli. It has been found that in order to efficiently express PspA, it is useful to truncate the mature PspA molecule of the Rx1 strain from its normal length of 589 amino acids to that of 314 amino acids comprising amino acids 1 to 314. This region of the PspA molecule contains most, if not all, of the protective epitopes of PspA. However, immunogenicity and protection studies in mice have demonstrated that the truncated recombinant form of PspA is not immunogenic in naive mice. Thus, it would be useful to improve the immunogenicity of recombinant PspA and fragments thereof. Moreover, it would be highly desirable to employ a pneumococcal antigen in a combination or multivalent composition. For instance, influenza (Flu) is a problematical infection, especially in the elderly and the young, as well as pneumonia; and, yearly Flu shots are common, especially in North America. Thus, it would be desirable to be able to administer Flu and pneumococcal antigens in one preparation.
Helicobacter pylori is the spiral bacterium which selectively colonizes human gastric mucin-secreting cells and is the causative agent in most cases of nonerosive gastritis in humans. Recent research activity indicates that H. pylori, which has a high urease activity, is responsible for most peptic ulcers as well as many gastric cancers. Many studies have suggested that urease, a complex of the products of the ureA and ureB genes, may be a protective antigen. However, until now it has not been known how to produce a sufficient mucosal immune response to urease without cholera toxin or related adjuvants.
Antigens or immunogenic fragments thereof stimulate an immune response when administered to a host. Such antigens, especially when recombinantly produced, may elicit a stronger response when administered in conjunction with adjuvant. Currently, alum is the only adjuvant licensed for human use, although hundreds of experimental adjuvants such as cholera toxin B are being tested. However, these adjuvants have deficiencies. For instance, while cholera toxin is a good adjuvant, it is highly toxic. On the other hand, cholera toxin B, while non-toxic, has no adjuvant activity. It would thus be desirable to provide immunological compositions capable of eliciting a strong response without the need for an adjuvant.
In certain instances when multiple antigens (two or more) are administered in the same preparation or sequentially, a phenomenon called efficacy interference occurs. Simply, due to the interaction of one or more antigens in the preparation with the host immunological system, the second or other antigens in the preparation fail to elicit a sufficient response, i.e., the efficacy of the latter antigen(s) is interfered with by the former antigen(s). It would thus be desirable to provide multivalent immunological compositions which do not give rise to this efficacy interference phenomenon; for instance, without wishing to necessarily be bound by any one particular theory, because the second antigen is a lipoprotein and as such is having an adjuvanting effect on the first antigen and, when in a combination composition with an adjuvant, a synergistic potentiating effect is obtained (whereby the first antigen is not interfering with the second antigen and vice versa).
More generally it would be desirable to enhance the immunogenicity of antigens by methods other than the use of an adjuvant, and to have the ability to employ such a means for enhanced immunogenicity with an adjuvant, so as to obtain an even greater immunological response.
Above-referenced U.S. Ser. No. 08/446,201 discloses that mucosal administration of killed whole pneumococci, lysate of pneumococci or isolated and purified PspA, as well as immunogenic fragments thereof, particularly when administered with an adjuvant, provides protection in animals against pneumococcal colonization and systemic infection. It has now been surprisingly found that mucosal administration of other antigens, such as urease, along with a lipoprotein, elicits systemic and local responses in animals without the use of an adjuvant.
It is believed that heretofore the art has not taught or suggested: immunological compositions comprising at least one antigen and a lipoprotein, and, optionally, an adjuvant, more preferably an antigen, an antigenic lipoprotein and, optionally, an adjuvant, and methods for administering the same as a multivalent composition, or for administering those components simultaneously or sequentially, especially such compositions and methods having enhanced immunogenicity.
It is an object of the invention to provide immunological compositions and methods for making and using the same.
It is a further object of the invention to provide immunological compositions having enhanced immunogenicity; or, compositions the administration of which potentiates the immunological response.
It is another object of the invention to provide methods for inducing an immunological response, preferably a potentiated response, involving administration to a suitable host such immunological compositions.
It is yet an additional object of the invention to provide an immunological composition comprising at least one antigen and at least one lipoprotein and, optionally, an adjuvant, preferably such compositions wherein the lipoprotein is antigenic.
It is still a further object of the invention to provide a method for inducing or potentiating an immunological response comprising administering to a host, animal or human, at least one antigen and at least one lipoprotein, and optionally, an adjuvant; and more preferably such methods wherein the lipoprotein is antigenic.
It has surprisingly been found that administration to a host of at least one lipoprotein with at least one antigen provides an immunological response by the host. The immunological response is generally better than that obtained by administration of the antigen alone.
Moreover, it has also surprisingly been found that administration to a host of at least one antigen, at least one lipoprotein and, optionally an adjuvant by either co-administration or by sequential administration (over a suitable time period such that each of the antigen, adjuvant and lipoprotein are present within the host at the same time) obtains an immunological response to the antigen by the host. This immunological response is generally better than that obtained by administration of the antigen alone or by administration of the antigen and adjuvant. Lipidated proteins appear to stimulate the immune response, in the manner of the adjuvant cholera toxin B.
Furthermore, it has additionally been surprisingly found that in these administrations the lipoprotein itself can be immunogenic or antigenic, e.g., be an antigen, and that not only is the immunological response to the antigen by the host obtained; but also, an immunological response to the antigenic lipoprotein is obtained. The immunological response to the antigenic lipoprotein can be as good as, or better than, that obtained by administration of the lipoprotein alone or with an adjuvant; and, the immunological response to the antigen can be better than that obtained by administering the antigen alone or the antigen and adjuvant.
The term lipoprotein as used herein is meant to exclude prior art lipopeptides; ergo, a lipoprotein can have more than 2 to 10 amino acids, or more than 18 to 20 amino acids, or greater than 24 amino acids, or 30 or more amino acids. Lipoproteins are larger molecules which reduce the amount of antigen and/or administrations of the antigen, despite a prejudice in the art against lipoproteins, e.g., Vaccine (1994) 12(14):1335, 1338 last line, column 1, to first line, column 2 (xe2x80x9csoluble proteins . . . not immunogenic [by oral routes]xe2x80x9d). Prior lipopeptides, due to their small size, can have at most one epitope whereas lipoproteins that can be used in the present invention can have more than one epitope, e.g. one B and one T, or can even be antigenic in their own right. Lipopeptides, in addition to being shorter and having less molecular weight than lipoproteins, and being difficult to synthesize because usually are made by Merrifield or other synthesis methods, differ from lipoproteins in that lipoproteins are larger, generally not made by Merrifield synthesis methods, and can be from isolation from natural sources or from recombinant techniques. That is, lipopeptides of the prior art were synthetically made, which limits their size to no more than about thirty amino acids. Lipoproteins are larger and of greater molecular weight than lipopeptides, and, unlike lipopeptides, are generally not made by Merrifield synthesis methods. Lipoproteins can be isolated from natural sources or produced by recombinant techniques. Further, lipoproteins are more soluble than lipopeptides. Additionally, peptides do not have quaternary or tertiary structure whereas proteins can have quaternary and/or tertiary structure. Based upon their ability to form tertiary structure, proteins have the ability to form functional domains which peptides cannot. Thus, there are several differences between prior xe2x80x9clipopeptidesxe2x80x9d and xe2x80x9clipoproteinsxe2x80x9d as used in this invention.
The lipoproteins formulations of the invention can be administered nasally and this is advantageous.
According to the present invention, it also has been found that a lipoprotein administered with an antigen according to the present invention is 500 times more potent then administration of a lipopeptide and an antigen.
Accordingly, the present invention provides an immunological composition comprising at least one antigen and at least one lipoprotein. The composition can further optionally, but not necessarily, comprise an adjuvant. Preferably the lipoprotein is an antigen. The immunological composition can be a vaccine.
The present invention further comprises a method for inducing an immunological response in a host comprising administering the aforementioned immunological composition. The method can be for inducing a protective response, e.g., when the immunological composition is a vaccine.
The present invention further comprises a method for inducing an immunological response comprising sequentially administering a first composition comprising an antigen, and a second composition comprising a lipoprotein. optionally either the first or second composition, or both the first and second compositions can further comprise an adjuvant. Preferably the lipoprotein is an antigen. The sequential administration should be undertaken over a suitable period of time whereby each of the antigen, lipoprotein and optional adjuvant is present at the same time in the host; and, such a time period can be determined by the skilled artisan, from this disclosure, without undue experimentation and by methods within the ambit of the skilled artisan, such as host sera titrations involving analysis thereof for the presence of antigen or antibody by, for instance, ELISA analysis. The administration may be mucosal, e.g., intragastric or intranasal.
The present invention particularly involves methods for inducing an immunological response in a host comprising the steps of mucosally administering to the host at least one antigen, and mucosally administering to the host at least one lipoprotein. The administration can be simultaneous or sequential. The antigen may be a bacterial protein or fragment thereof, e.g. urease.
The xe2x80x9cantigenxe2x80x9d in the inventive compositions and methods can be any antigen to which one wishes to elicit an immunological response in a host, animal or human. For instance, without wishing to necessarily limit the invention, the antigen can be: a Borrelia antigen, e.g., OspA, OspC, OspB, OspD; a pneumococcal antigen, e.g., PspA; an influenza (Flu) antigen such as HA; a pertussis or whooping cough antigen such as the pertussis 69KD polypeptide; a hepatitis antigen, e.g., hepatitis B antigen such as hepatitis B surface antigen; a Helicobacter pylori antigen such as urease; a rabies virus antigen, e.g., rabies G antigen; a flavivirus antigen, e.g., a Japanese encephalitis virus, Dengue virus or yellow fever virus antigen; a chicken pox virus antigen; a diphtheria antigen; a C. tetani antigen, e.g., tetanus toxoid; a mumps virus antigen; a measles virus antigen; a malaria antigen; a herpes virus antigen, such as an alphaherpesvirus, betaherpesvirus or gammaherpesvirus antigen, e.g., a herpes virus glycoprotein, for instance an equine herpesvirus antigen, e.g., gp13, gp14, gD, gp63, or gE, a pseudorabies virus antigen, e.g., gp50, gpII, gpIII, gpI, a herpes simplex virus antigen, e.g., gC, gD, a bovine herpes virus antigen, e.g., gI, a feline herpes virus antigen, e.g., gB, an Epstein-Barr virus antigen, e.g., gp220, gp340, or gH, or a human cytomegalovirus antigen, e.g., gB; a human immunodeficiency virus antigen, e.g., gp160 or gp120; a simian immunodeficiency virus antigen; a bovine viral diarrhea virus antigen; an equine influenza virus antigen; a feline leukemia virus antigen; a canine distemper virus antigen, e.g., HA or F glycoproteins; a canine adenovirus antigen, e.g., canine adenovirus type 2 antigen; a canine coronavirus antigen; a canine parainfluenza antigen; a canine parvovirus antigen; a Hantaan virus antigen; an avian influenza virus antigen e.g., a nucleoprotein antigen; a Newcastle Disease virus antigen, e.g., F, HN; an antigen of rous associated virus, e.g., an RAV-1 envelope antigen; an infectious bronchitis virus antigen, e.g., a matrix antigen or a preplomer antigen; an infectious bursal disease virus antigen; a cholera antigen; a tumor associated antigen; a feline immunodeficiency virus antigen; a foot-and-mouth disease virus antigen; a Marek""s Disease Virus antigen; a Staphylococci antigen; a Streptococci antigen; a Haemophilus influenza antigen, e.g., group b polysaccharide-protein conjugates; a papilloma virus; a poliovirus antigen; a rubella virus antigen; a poxvirus, such as smallpox antigen, e.g., vaccinia; a typhus virus antigen; a typhoid virus antigen; a tuberculosis virus antigen; an HTLV antigen; or, other bacteria, virus or pathogen antigen, such as a bacterial or viral surface antigen or coat protein.
The antigen can be a known antigen; can be isolated from the bacteria, virus or pathogen; or, can be a recombinant antigen from expression of suitable nucleic acid coding therefor by a suitable vector, and isolation and/or purification of the recombinant antigen. The selection of the antigen is, of course, dependent upon the immunological response desired and the host.
The lipoprotein can be any lipoprotein which is compatible physiologically with the host. Most preferably it is a bacterial lipoprotein or a lipoprotein having a bacterial lipid moiety.
The lipoprotein is preferably itself also an antigen. Thus, the lipoprotein is preferably an outer membrane component of a pathogen, e.g., virus or bacteria, more preferably a lipoprotein which has an extrinsic or peripheral protein such that the lipoprotein is extracted with mild conditions or detergent without substantial denaturation or loss of lipid moiety (so as to retain epitopes). However, any antigenic lipoprotein can be employed in the practice of the invention. And, the lipoprotein can be isolated from a suitable physiological source, or from an organism, e.g., bacteria; or can be recombinantly produced. Thus, the lipidated Borrelia antigens, e.g., recombinant OspA, and, the lipidated OspA and Borrelia fractions containing lipidated proteins (isolated by mild conditions) disclosed in the applications referenced in the Reference to Related Applications, and in WO 90/04411 (incorporated herein by reference) can be used as the lipoprotein in the practice of the invention. Of course, the xe2x80x9cantigenxe2x80x9d and the xe2x80x9clipoproteinxe2x80x9d in the invention are separate, different ingredients (such that, for instance, when the xe2x80x9clipoproteinxe2x80x9d is OspA, it is not also the xe2x80x9cantigenxe2x80x9d).
In application Ser. No. 08/475,781 filed Jun. 7, 1995 and incorporated herein by reference, recombinant lipoproteins, especially antigenic recombinant lipoproteins, for instance, those from expression of the leader sequence of OspA for the lipidation thereof, are disclosed; and, those recombinant lipoproteins may be employed in the practice of the invention. As to expression of recombinant proteins, it is expected that the skilled artisan is familiar with the various vector systems available for such expression, e.g., bacteria such as E. coli and bacterial viruses, and the like.
The adjuvant can be any vehicle which would typically enhance the antigenicity of the antigen, e.g., a suspension or gel of minerals (for instance, alum, aluminum hydroxide or phosphate) on which the antigen is adsorbed; or a water-in-oil emulsion in which antigen solution is emulsified in mineral oil (e.g., Freund""s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (e.g., Freund""s complete adjuvant); or cholera toxin (sometimes with cholera toxin B, which may enhance the effect); or, any of the other adjuvants known in the art, or discussed in the Background of the Invention. The antigen and/or the lipoprotein can be absorbed onto or coupled with the adjuvant.
Presently preferred embodiments of the invention involve: alum as the adjuvant if an adjuvant is present; OspA, or a recombinant OspA leader/PspA, a recombinant OspA leader/OspC, a recombinant OspA leader/UreA of H. pylori, or, a recombinant OspA leader/UreB of H. Pylori as the lipoprotein (OspA leader/PspA is a recombinant lipoprotein having a lipidated moiety from expression of the OspA leader nucleic acid sequence and a protein moiety from expression of a pspA nucleic acid sequence; OspA leader/OspC is analogous to OspA leader/PspA, except that the protein moiety is from expression of an ospc nucleic acid sequence and OspA leader/ureA and OspA leader/ureB are also analogous to OspA leader/PspA, except that the protein moiety is from expression of a ureA or ureB nucleic acid sequence); and ospc or another Borrelia antigen, or an influenza antigen, e.g., HA (such as from influenza A, e.g., Texas strain), or urease as the antigen. Particular embodiments can include compositions: (i) comprising alum [adjuvant], OspA [lipoprotein] and another Borrelia antigen such as OspC [antigen]; (ii) comprising alum [adjuvant], OspA [antigen], and OspA leader/OspC [lipoprotein]; (iii) comprising alum [adjuvant], OspA leader/PspA [lipoprotein] and influenza antigen, e.g., influenza A HA [antigen] (iv) OspA [lipoprotein] and an H. pylori antigen, e.g., urease [antigen].
Other objects and embodiments of the invention are disclosed in or are obvious variants from the following description.